Tumor necrosis factor-? (TNF?) has potent, direct antiviral activity but has not been exploited as an antiviral therapy because of its systemic toxic effects. We have observed that primary human liver cells and hepatoma cells treated with low levels of TNF? are resistant to infection with the hepatitis C virus (HCV) in vitro. This effect is dependent on TNF? interaction with its receptor and is seen in the absence of detectable IFN? or IFN? gene expression. Sindbis virus, an alphavirus, infects Huh-7 hepatoma cells and is insensitive to TNF? pretreatment. However, TNF? synergizes with IFN? to block Sindbis virus infection. We propose to use state-of-the-art techniques to identify key mediators of TNF?'s antiviral effects alone and in combination with IFN?. We propose three Specific Aims. In the first Aim, we will test the requirements for signal transduction through the major pathways activated by TNF? binding to its receptor, using pharmacologic inhibitors as well as dominant negative regulators of TNF? signal transduction. In the second Aim, we will use novel virological and cell biological tools to define the stages in HCV infection that are inhibited following TNF? treatment. In the third Aim, we will use microarray and next-generation sequencing approaches to define the effects of TNF? on the transcriptome, identify candidate genes that may mediate the development of an antiviral state in TNF?- treated cells, and test the importance of these genes by knockdown and overexpression studies. The long-term goals of this research are to identify specific cellular pathways that mediate TNF?'s antiviral activity, and to determine whether it is possible to isolate these activities from TNF?'s systemic toxic effects.